Project Summary/Abstract In the cell nucleus, the three-dimensional (3D) folding of the genome regulates many genomic functions, ranging from gene expression regulation to DNA replication, recombination, and repair. 3D genomic structures exist at a variety of length scales, and changes in genome structures are known to be associated with cancer, but a true physical picture of genome folding in cancer cells within their heterogeneous tumor microenvironment is still elusive. It is also unknown how variations of the 3D genome organization may affect single-cell gene expression in tumors. Existing sequencing-based omics approaches cannot address these questions due to technical limitations. Here we propose the advanced development and validation of a new imaging method that can directly trace the spatial folding of the genome across multiple length scales and image numerous RNA species with single-molecule resolution in the same single cells in heterogeneous tumors. We will test this method with mouse tumor models that enable lineage tracing of cancer clones during progression and with clinical samples from human patients. We expect this technologic advance to transform 3D genome investigation in cancer biology and lead to new biomarkers for cancer diagnosis, prognosis, and treatment.